Screening process for atopic dermatitis

ABSTRACT

The present invention provides a process of screening patients for atopic dermatitis. The process includes the step of determining, in sera of the patient, the presence of antibodies against nuclear antigens such as transcription co-activator p75, wherein the presence of such antibodies indicates atopic dermatitis. The screening process can be used to detect atopic dermatitis in patients suffering from other conditions such as asthma or interstitial cystitis.

Funds used to support some of the studies reported herein were providedby the National Institutes of Health (NIH grants DK49413 and AR32063).The United States Government may, therefore, have certain rights in anyinvention disclosed herein.

TECHNICAL FIELD OF THE INVENTION

The field of this invention is diagnosis of atopic dermatitis.

BACKGROUND OF THE INVENTION

Atopic dermatitis (AD) is a chronic, relapsing, pruritic skin disorderthat generally first appears in childhood and frequently progresses toasthma and/or allergic rhinitis in the adult (Hanifin et al., ActaDermatovener (Stockholm) Suppl 1980; 92:44-47; Leung et al., Dermatologyin General Medicine, 4th edition, 1993, pp. 1543-1564; Cooper, J InvestDermatol 1994; 102:128-137). There are no reliable laboratory markersfor this condition but AD patients often have elevated serum IgE levels,allergic reactivity to foods and to other common allergens such aspollens, molds, and insects. There have also been reports of antinuclearantibodies (ANAs) in this condition (Taniguchi et al., Acta DermVenereol (Stockh) Suppl 1992; 176:62-64; Tada et al., Dermatol 1994;189:38-40). The prevalence of AD appears to be on the rise, with 10-15%of the population being affected at some time during childhood(Beltrani, J Allergy Clin Immunol 1999; 104:587-598; Leung, J AllergyClin Immunol 1999; 104: S99-S108). We now report the finding of anautoantibody-autoantigen system in 30% of patients with AD which is alsoshared to a lesser extent by patients with asthma and interstitialcystitis (IC). IC is a urinary bladder condition in which the classicalpathology is characterized by predominant mononuclear cell infiltrationof the lamina propria with lymphocytes, plasma cells and mast cells(Gillenwater, et al., J Urol 1988; 140:203-206).

BRIEF SUMMARY OF THE INVENTION

The present invention provides a process of screening patients foratopic dermatitis. The process includes the step of determining, in seraof the patient, the presence of antibodies against nuclear antigens suchas transcription co-activator p75, wherein the presence of suchantibodies indicates atopic dermatitis. The screening process can beused to detect atopic dermatitis in patients suffering from otherconditions such as asthma or interstitial cystitis.

In accordance with the process, sera is obtained from the patient andcontacted with the nuclear antigen (e.g., nuclear transcriptionco-activator p75). The resulting mixture is maintained for a period oftime sufficient for formation of an immune complex between antibodies inthe sera and the antigen. The antibodies can be of the IgG or IgE classof immunoglobulins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (shown in two panels designated 1A and 1B) shows the cloning andsequence data for DFS70. Panel 1A is a schematic representation ofoverlapping cDNAs. Clone 6.1 was obtained by immunoscreening a T24 cDNAlibrary with a human anti-DFS serum and clone 52 by 5′ RACE. Thecombined cDNA represented 3117 nucleotides. Panel 1B shows thenucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID NO:1) sequencesof DFS-70. Sequencing of both DNA strands was performed with customsynthetic oligonucleotide primers. The open reading frame starts atnucleotide 54 and ends at nucleotide 1645. The methionine start sequenceand the polyadenylation signals AATAAA/ATTAAA are underlined. Thecombined nucleotide and amino acid sequences for the original clone 6.1,starting at nucleotide 593 and ending at nucleotide 3117, were submittedto GenBank under accession number U94319 in March 1997. The differencesin nucleotides between DFS70 and p75 (residues single underlined) are atposition 726 a→t (Glu→Gly) and 1314 a→t (Tyr→Phe) while there were nodifferences between DFS70 and LEDGE (lens epithelium derived growthfactor) at these positions. DFS70 differed from both p75 and LEDGF(residues double underlined) at position 188 g→t (Val→Phe), 244 g→a (noaa change) and 347 a→g (Thr→Ala).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process of screening patients foratopic dermatitis. The process includes the step of determining, in seraof the patient, the presence of antibodies against nuclear antigens suchas transcription co-activator p75, wherein the presence of suchantibodies indicates atopic dermatitis. The screening process can beused to detect atopic dermatitis in patients suffering from otherconditions such as asthma or interstitial cystitis.

The identification of an antigen-antibody system in AD was initiated byan observation in IC patients of an autoantibody against a nuclearantigen distributed as dense fine speckles in the nucleoplasm of cellsin interphase and increased localization in condensed chromosomes inmitosis. In addition, in immunoblotting using tissue culture extract asthe antigen source, sera of such patients reacted with a 70 kilodalton(kDa) protein. This antigen was called DFS-70, incorporating theimmunolocalization of the antigen (dense fine speckles) and theestimated molecular size of the antigen in polyacrylamide-SDS gels (Ochset al., J Urol 1994; 151:587-592). The antibody was present in higherfrequency in AD patients than in IC patients. This antigen is a nuclearprotein, which is identical to a nuclear transcription co-activatorcalled p75. The antibody is also present in other conditions and it islikely that the underlying common feature is the presence of AD inpatients with asthma, interstitial cystitis and other disease states.

In accordance with the process of this invention, sera is obtained froma patient suspected on having AD. The sera is contacted with the nuclearantigen (e.g., nuclear transcription co-activator p75). The resultingmixture is maintained for a period of time sufficient for formation ofan immune complex between antibodies in the sera and the antigen. Theantibodies can be of the IgG or IgE class of immunoglobulins. Means forcollecting sera from patients are well known in the art. Determinationof immune complex formation can be accomplished by any means known inthe art. A specific example of such determination is set forth hereinbelow.

The following teaching discloses a particular embodiment of thisinvention. A skilled artisan will readily recognize that otherembodiments can be used. Thus, the particular teachings set forth beloware not limiting of the specification and claims in any way.

Patients and Antibody Controls

Sixty-four patients with atopic dermatitis, from the Department ofDermatology, Nagoya University School of Medicine, Nagoya, JAPAN, wereenrolled in our study. Diagnostic criteria for AD were those describedby Hanifin and Rajka (Hanifin et al., Acta Dermatovener (Stockholm)Suppl 1980; 92:44-47). Chart records were reviewed for age, sex,duration of disease, eosinophil numbers, IgE levels, presence ofrespiratory atopy (bronchial asthma, allergic rhinitis), and a degree offacial dermatitis. Patients with interstitial cystitis were diagnosedusing established criteria (Gillenwater et al., J Urol 1988;140:203-206) and were part of a large study on clinical features andsero-epidemiology reported previously (Koziol et al., J Urol 1993;149:465-469; Ochs et al., Interstitial Cystitis 1997, pp.47-52).

Patients with asthma were recruited from the Division of Allergy andImmunology, Scripps Clinic, La Jolla and fulfilled the American ThoracicSociety criteria for the diagnosis of asthma. Psoriasis patients withoutaccompanying atopic dermatitis came from a clinical practice. Patientswith chronic fatigue syndrome were reported and characterized in aprevious study (Konstantinov et al., J Clin Invest 1996; 98:1888-1896).Normal human sera and sera from patients with systemic lupuserythematosus, rheumatoid arthritis, Sjögren's syndrome, and sclerodermawere obtained from the serum bank of the W. M. Keck Autoimmune DiseaseCenter, Scripps Research Institute, La Jolla, Calif.

Immunofluorescence

Sera from patients and controls were diluted 1/100 in PBS and examinedby indirect immunofluorescence for the presence of autoantibodies tonuclear and cytoplasmic antigens on commercially prepared HEp-2 cellsubstrate slides (Bion, Park Ridge, Ill.) with anti-human IgG coupled toFITC (Caltag, San Francisco, Calif.) as the secondary detecting reagent.IgG subclass identification of autoantibodies was performed by indirectimmunofluorescence on commercially prepared HEp-2 cell substrate slidesas described above using affinity-purified FITC-conjugated sheepanti-human IgG1, IgG2, IgG3, and IgG4 (The Binding Site, Birmingham,England) as secondary antibodies.

Immunoblotting

Human MOLT-4 cells (a T cell line) were obtained from the American TypeCulture Collection (Rockville, Md.) and grown in suspension culture inRPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and10 μg/ml gentamicin sulfate. Whole-cell extracts were prepared fromMOLT-4 cells by pelleting suspended cells in media, rinsing in PBS, andresuspending the cell pellet in an equal volume of 2× gelelectrophoresis sample buffer containing a cocktail of proteaseinhibitors (catalog #1697498, Boehringer Manmheim, Indianapolis, Ind.).The cell extract was then sonicated on ice for 4-5 bursts of 10 sec.each until the cells were dispersed. After sonication, the extract wasthen passed through progressively finer gauge needles, from #18 to #27,to shear the DNA and then the extract was boiled for 5 min., centrifugedto remove insoluble material, and aliquots stored at −70° C. This formof cell extract has been used extensively in many previous studies (Ochset al., Interstitial Cystitis 1997, pp.47-52; Konstantinov et al., JClin Invest 1996; 98:1888-1896) as a source of autoantigens fordetection of serum autoantibodies against ubiquitous cellular antigens.

Western blotting was performed essentially as described by Chan andPollard (Chan et al., Manual of Clinical Laboratory Immunology 1992,pp.755-762). Serum was diluted 1/100 and detection of immunoreactivebands was performed by chemiluminescence using peroxidase-conjugatedgoat anti-human IgG (Caltag, Burlingame, Calif.) and the ECLchemiluminescence kit (Amersham, Arlington Heights, Ill.) according tothe manufacturer's directions, followed by autoradiography. For blottingof recombinant DFS70, the detection procedure was identical.

For detection of specific IgE autoantibodies to DFS70, serum sampleswere diluted 1/10 according to Valenta et al. (Valenta et al., J InvestDermatol 1996; 107:203-208) in order to detect serum IgE levels that areon average 100,000 times lower in amount compared to levels of IgG. IgEantibody was detected using peroxidase-conjugated goat IgG antibody tohuman IgE epsilon heavy chain (Cappel, Durham, N.C.) as the secondarydetecting reagent, followed by chemiluminescence and autoradiography asdescribed above for the detection of IgG. Normal control sera wereincluded in every determination in order to exclude spurious findings intlis procedure.

cDNA Cloning

DFS70 was originally described using autoantibodies from patients withinterstitial cystitis (Ochs et al., J Urol 1994; 151:587-592) and wasgiven the designation because of a characteristic immunohistochemicalstaining pattern on HEp-2 (human epithelial Line) cells consisting ofdense fine specldes distributed in the nucleoplasm in interphase cellsand with accentuated generalized staining of condensed chromosomes inmitotic cells. These sera also showed reactivity with a 70 kDa proteinin MOLT-4 extracts in Western blotting. One of these sera, which washigh in antibody titer and available in larger quantity, was used toisolate a partial cDNA sequence as indicated below.

The method for primary screening of a T24 (bladder carcinoma cell line)cDNA expression library was reported previously (Ochs et al., Molec BiolCell 1996; 7:1015-1024). Briefly, serum from patient 90-49 was diluted1/100 and preabsorbed with wild-type λ Zap Express phage withoutinserts. The preabsorbed serum was then used to screen approximately300,000 recombinant plaques from a cDNA expression library constructedfrom T24, a bladder epithelial cell line (ATCC, Rockville, Md.), using¹²⁵I-staph protein A according to the method of Young and Davis (Younget al., Proc Natl Acad Sci (USA) 1983; 80:1194-1198). All screeningswere carried out on duplicate filters and two double-positive cloneswere obtained. After multiple rescreening, clone DFS6.1 was amplified,purified, and used for sequence analysis. Clone DFS6.1 was a partialcDNA and in order to isolate full-length cDNA multiple overlapping 5′clones were obtained from human placenta cDNAs using a modified 5′-RACEmethod previously described (Lung et al., Trends Genet. 1996;12:389-391). Clone DFS6.1 was reported in an abstract (Ochs et al.,Molec Biol Cell 1995; 6:75a Suppl.) and the nucleotide sequence wassubmitted to Gen Bank under accession No. U94319 in March 1997.

Sequence and Protein Anaylses

cDNA inserts were analyzed by restriction mapping and sequencing.Nucleotide sequence was determined by dye primer cycle sequencing usinga Model 373A DNA sequencer from Applied Biosystems (ABI, Foster City,Calif.). Oligonucleotide primers were synthesized with a Model 394 DNAsynthesizer (ABI). DNA and protein sequences were analyzed by theGenetics Computer Group (GCG) Sequence Analysis Software Package version7.2 for UNIX computers (Deveraux et al., Nucleic Acids Res 1984;12:387-395). Alignment of protein sequences was achieved with the GAPprogram that employed the algorithm of Needleman and Wunsch. Molecularmass was calculated using the GCG program PEPTIDESORT.

Purification of Recombinant DFS70

For expression and purification of recombinant protein, the EcoR1-EcoR1fragment of clone DFS6.1 and the fall-length cDNA were subcloned intopET 28 vector (Novagen, Madison, Wis.). The full-length cDNA wasobtained by RT-PCR using T24 cell mRNA and primers corresponding tosequences flanking the methionine start codon and 3′ stop codon. Thesense and antisense primers were 5′-GCAGAATTCGATACATGACTCGCGATTTC-3′(SEQ ID NO:3) and 5′-AGTGAAGCTTATATTCCAGGTATGTCAACCTACGTT-3′ (SEQ IDNO:4) and the sequences incorporated to facilitate subdloning were EcoRIand HindIII restriction sites. The pET28 vector has N-terminal fusiontags comprising T7 and 6× histidine. The T7 fusion tag allows for easydetection of recombinant protein with T7 antibodies and the 6 xhistidines allow for protein purification due to the high affinity ofhistidine to nickel. E. coli strain BL21-DE3 containing the pET 28avector with the DFS6.1 insert was grown in LB media containing kanamycinuntil OD₆₀₀=0.8. Then, 5 mM IPTG was added to induce recombinant proteinexpression for 6 hrs. at 37° C. Cells were harvested by centrifugationand broken open by cycles of freezing and thawing in a buffer solutioncontaining 6M guanidine hydrochloride according to the manufacturer'sdirections (Qiagen, Chatsworth, Calif.). This solution was then passedthrough a Ni-NTA-Agarose resin (Qiagen, catalog #30210) column where therecombinant protein is bound at elevated pH (pH 8.0) by the affinity ofnickel for the 6 x His portion, unbound material washed out, and therecombinant protein eluted in fractions at a pH of 6.0. Each fractionwas checked by Western blotting for the presence and purity ofrecombinant protein.

Recombinant Proteins and Antibodies for Transcription Coactivators p52and 75

The USA (upstream stimulatory activity) fraction of HeLa cellscontaining the transcription coactivator proteins p52 and p75 wasdescribed previously (Ge et al., Cell 1994; 78:513-523). Subsequentstudies resulted in the cDNA cloning and production of recombinantproteins for p52 and p75 and the production of rabbit polyconalantibodies (Ge et al., EMBO J. 1998; 17:6723-6729). These reportsappeared while these studies were in progress and it was noted that ourplasmid DFS 6.1 had complete identity with the corresponding region ofp75. Studies were therefore initiated with Ge and colleagues (NIH,Bethesda, Md.) and reagents exchanged to determine the reactivity ofautoantibodies to p75 and p52 as described below.

Immunoelectron Microscopy

Small pieces of mouse intestine were fixed for 1 hr. at 4° C. with 1%glutaraldehyde buffered in PBS. Pieces were then dehydrated in 70%ethanol and embedded in LR White. After polymerization at 56° C. for 2days, sections were collected grids. For immunolabeling, individualgrids (with sections down) were incubated for 30-60 min. with a blocldngsolution consisting of 2% NGS/0.2% BSA made in Tris-buffered saline(TBS) containing 1.0% Tween-20 (0.02 M Tris, 0.15 M Na Cl, 1.0%Tween-20, pH 7.6). Grids were then transferred to drops consisting of a1/100 dilution of human anti-DFS70 made in TBS/Tween-20 for 1 hr. at R.T. and then rinsed with TBS/Tween-20 10×1 min. each. Grids were thenblocked for 30-60 min. in TBS/Tween-20 and incubated for 1 hr. at R. T.in a 1/50 dilution of goat anti-human IgG linked to 10 nm colloidal gold(Amersham) diluted in TBS/Tween-20. Grids were then rinsed inTBS/Tween-20 10×1 min. each, rinsed in PBS, fixed 30 min. at R. T. in 1%glutaraldehyde/PBS, rinsed in PBS 3×5 min., rinsed in distilled water3×5 min, counterstained with uranyl acetate and lead citrate, rinsed indistilled water, and blotted dry.

ANAs in Patients with Atopic Dermiatitis

Sixty-four AD patients, 30 males and 34 females, were analyzed for IgGautoantibodies to nuclear antigens (abbreviated as antinuclear antibody[ANA]) by indirect immunofluorescence on commercial human HEp-2 cellsubstrates and by Western blotting on whole-cell extracts of humanMOLT-4 cells (FIG. 1). AD patients ranged in age from 4-43 yrs. (averageof 24.4 yrs.) and the duration of disease ranged from 1-35 yrs. (average13.4 yrs.). Of these 64 AD patients, 26 (40.6%) had ANAs of one patternor another but only 18 (28%) produced a pattern of dense fine-specklesin interhase nucleoplasm and staining of chromosomes in mitotic cells.

TABLE 1 Clinical and Laboratory Data on 18 Atopic Dermatitis Patientswith DFS70-Staining Pattern in Immnunobistochemistry AntinuclearAntibodies Patient Sex Age^(a) Dur.^(b) Eos^(c) IgE^(d) R.A.^(e)F.D.^(f) Pattern^(g) Titer^(h) Anti-DFS70^(i) 6 F 20 15 ND ND BA/AR yesDFS/chr 1280 pos 8 F 4 2 400 67 no yes DFS/chr >1280 pos 10 F 23 22 2891859 No Yes DFS/chr 320 pos 12 M 19 14 1463 13226 BA/AR yes DFS/chr/NBs640 pos 13 F 20 16 659 4224 BA/AR yes DFS/chr 640 pos 15 F 29 14 1440 65no yes DFS/chr/No >1280 pos 16 F 22 13 566 6900 no yes DFS/chr 320 neg28 M 23 23 629 5024 AR yes DFS/chr 320 pos 36 F 20 6 213 488 AR yesDFS/chr 1280 pos 38 F 15 7 743 2189 no yes DFS/chr 320 pos 39 F 20 201296 6270 AR yes DFS/chr 1280 pos 41 F 19 9 885 196 no yes DFS/chr/NBs640 pos 68 F 20 8 568 1579 no yes DFS/chr 640 pos 77 F 27 26 975 1891 nono DFS/chr ND neg 78 M 21 9 87 1714 no yes DFS/chr ND neg 81 M 26 10 20525 no no DFS/chr 320 pos 88 F 19 3 300 84 AR yes DFS/chr/NBs 640 pos 99F 32 27 152 272 AR yes DFS/chr 640 neg ^(a)Age in years ^(b)Duration ofdisease in years ^(c)Eosinophils/ml of blood ^(d)IgE levels in IU/ml ofblood ^(e)Respiratory Atopy - BA, broncial asthma; AR, allergic rhinitis^(f)Facial dermatitis ^(g)DFS, dense fine speckled staining; chr,mitotic chromosome staining; NBs, staining of unidentified nuclearbodies; No, nucleolar staining ^(h)Titer of ANA; ND, not done^(i)Western blotting against recombinant DFS70; pos, positive; neg,negative

The prototypic immunofluorescence staining pattern AD sera shows thatinterphase cell nuclei display fine specldes distributed in thenucleoplasm and aggregation of the specldes in the region of condensedchromosomes hi mitotic cells. A prototypic IC serum showed a similarpattern. Thirteen of the 18 patients described in Table 1 were includedin the Western blot analysis depicting the initial studies, which showedthat the majority of patients with DFS type antibodies appeared to bereacting with an antigen of approximately 70 kDa contained in MOLT-4cell extracts. Lanes 5 to 22 consisted of 13 sera from patients in Table1 with the addition of 5 other AD patients who did not have thedistinctive DFS type of autoantibodies and other control positive andnegative sera in lanes 1 to 4. It was noted that some AD sera hadantibodies to antigens of other molecular sizes but the importantfinding was the occurrence of a common antigen-antibody system in themajority of sea with DFS-type antibodies. The last column of Table 1includes data on the 18 DFS sera with respect to antibodies torecombinant antigen which is described firrther below. It should benoted from Table 1 that there was no significant relationship betweenthe presence anti-DFS-70 antibody and parameters of age, duration ofdisease, eosinophils and IgE levels or with respiratory atopy but themajority of patients had facial dermatitis.

cDNA Cloning of DFS70: Sequence Identity to tite TranscriptionalCo-activator p75

To characterize DFS70 and to determine whether or not it was a novelprotein recognized by the sera of both AD and IC patients, we used themore abundant serum from IC patient 90-49 to clone the partial cDNA ofDFS70. We first isolated poly A(+) RNA from human T24 bladder epithelialcells and from this starting material a cDNA expression library wasconstructed, screened with serum, and the positive clones obtained weresubeloned and purified to homogeneity. Clone DFS6.1, containing a 2 kbinsert, was then cloned and sequenced (FIG. 1A). Analysis of thissequence showed an open reading frarne (ORF) of approximately 1 kb,coding for 351 amino acids with a predicted molecular weight of 40 kDa(FIG. 1B). A search of the gene sequence database at that time indicatedno identity with previously reported sequences. The sequence of cDNAclone DFS6.1 from protein DFS70 was deposited in GenBank under accession#U94319 (Ochs et al., Molec Biol Cell 1995; 6.75a Suppl.) in 1997.

An overlapping clone #52 was obtained from human placental cDNAs using5′RACE (FIG. 1A). Independent confirmation was provided from RT-PCRusing primers upstream of the methionine start and translation stopcodons. The amplified cDNA was subdloned into pET28 for sequencinganalysis as described herein above. The complete cDNA and deduced aminoacid sequences are shown in FIG. 1B. GenBank search at this time showedthat DFS70 was identical to transcription coactivator p75 (Ge et al.,EMBO J 1998; 17:6723-6729) and lens epithelium-derived growth factor(LEDGF) (Singh et al., Invest Ophthalmol Vis Sci 1998; 39:2132-9). Thetwo to four amino acid differences among these proteins (see FIG. 1B)may reflect errors in reverse transcriptase function during the cDNAsynthesis step for establishing the cDNA expression libraries sincethese are single nucleotide differences. It should be noted thatdifferent cell lines, T24 in this report, HeLa (Ge et al., EMBO J. 1998;17:6723-6729) and lens epithelial cell (Singh et al., Invest OphthalmolVis Sci 1998; 39:2132-9 (Abst)) cDNA libraries were used for isolatingthe different cDNA clones.

Western Blotting: A Shiared Autoantibody/Autoantigen System in AD and ICPatients

For a higher level of protein expression and to purify the recombinantprotein away from contaminating bacterial proteins that might interferein Western blotting, the full-length cDNA clone of DFS70 was excised andcloned into pET vector 28a (pET-DFS) that has a 5′ T7 fusion protein tagand a 6× histidine tag. Recombinant DFS70 protein expressed from clonepET-DFS was bound and specifically eluted from a nickel affinity columnand was detected with either T7 antibody or the serum from IC patient90-49 used to originally isolate clone 6.1 of DFS70. Purifiedrecombinant DFS70 was then used to confirm the presence of IgGantibodies originally detected using MOLT-4 cell extracts. Patients thatwere positive in Western blotting against a 70 kDa protein contained inMOLT-4 cells and were also positive against recombinant DFS70. All 64 ADsera were subsequently examined in Western blotting against recombinantDFS70 and 19 (29.6%) were positive (see Table 2).

TABLE 2 Patient Groups Reactive with Recombinant DFS70 by WesternBlotting Patients Reactivity Normal Controls 0/39 (0%) Atopic Dermatitis19/64 (29.6%) Asthma 8/50 (16%) Interstitial Cystitis 9/103 (8.7%)Psoriasis 1/22 (4.5%) Chronic Fatigue Syndrome 2/60 (3.3%) SystemicLupus Erythematosus 0/36 (0%) Rheumatoid Arthritis 0/30 (0%) Sjögren'sSyndrome 2/29 (6.9%) Scleroderma 1/40 (2.5%)

In summary, of 18 sera showing putative DFS staining patern byimmunohistochemistry, 14 were positive in Western blotting againstrecombinant DFS70 and of the 46 AD sera without this pattern inimmunohistochemistry, 5 were positive in Western blotting.

The IgG subclass was determined for 16 AD sera that were reactive forDFS70. All antibodies were of the IgG1 subclass and none were IgG4. Twosera also contained antibodies of the IgG2 subclass and 3 otherscontained antibodies of the IgG3 subclass.

Detection of IgE Autoantibodies to DFS70

Since blood levels of IgE are approximately 100,000 times less thanthose for IgG, we performed IgE Western blotting at serum dilution of1/10 compared to normal dilutions of 1/100 for IgG, and the signalsobtained also necessitated much longer autoradiographic exposure times(20 minutes for IgE antibody versus 5 seconds for IgG antibody). In bothIC and Ad patients we could detect specific IgE autoantibodies to DFS70by Western blotting. It was noted that in general, there was a positiverelationship between signals for IgG and for IgE antibodies in that astrong signal for IgG antibody correlated with a strong signal for IgEantibody.

Other Patient Groups Reactive with Recombinant DFS70

Table 2 summarizes the results of Western blotting against recombinantDFS 70 protein in various groups of patients and normal controls. Noneof the normal healthy controls were positive for IgG autoantibodiesreactive with recombinant DFS70 compared to 30% of AD patients. Thisresult compares with a positivity of 16% for asthma patients, 8.7% forIC, 4.5% for psoriasis patients, 3.3% for patients diagnosed withchronic fatigue syndrome, 0% for lupus patients, 0% for rheumatoidarthritis patients, 6.9% for Sjögren's patients, and 2.5% for patientswith scleroderma.

Human Autoantibodies to DFS70 Bind Transcription Coactivator p75, butnot p52

During the course of the studies described above, a report by Ge et al.(Ge et al., EMBO J. 1998; 17:6723-6729) described the isolation of cDNAsencoding transcription coactivators p52 and p75, which these authorsshowed were required for transcriptional activation in human cell-freesystems containing RNA polymerase It and general initiation factors. p52is a protein of 333 aa and is a splice variant of a gene that alsoencodes a protein p75 of 530 aa with the additional amino acids of p75located in the C-terminal region. Our initial DFS6.1 partial cDNA cloneencoded for the C-terminal 351 aa of p75 and it was of interest todetermine how AD sera reacted with the splice variants p75 and p52. Fivehuman sera, four from patients with atopic dermatitis (LB, KK,CK, WS)and an autoirnmune serum containing autoantibodies to other cellularantigens were coded and analyzed for reactivity in the Ge laboratorywith the upstream stimulatory activity (USA) fraction of HeLa cellswhich contained both p52 and p75. All the atopic dermatitis sera reactedwith p75 but not with p52 whereas the irrelevant autoimmune serum AFreacted with USA fraction to give two unrelated bands of unknownidentity. Rabbit antibodies raised against p52 and p75 fractions wereused as positive controls: anti-p52 reacted with both p52 and p75whereas anti-p75 reacted with p75 alone. This could be expected sinceanti-p52 was raised with full-length p52 as immunogen whereas anti-p75was raised with the unique C-terminal region of p75. The human sera werefurther analyzed in Western blotting with recombinant p75 and p52, withthe results confirming the observations made with the USA fraction.

Rabbit Antibodies to Recombinant p75 and p52 Bind to Recombinant DFS70and Display DFS Localization

It was demonstrated that rabbit antibodies raised against recombinanttranscription factors p75 and p52 were both able to bind to recombinantfull-length DFS70. When the rabbit antibodies were used inimmunohistochemistry, they displayed identical patterns ofimmunolocalization as compared to human anti-DFS70. A pattern of densefine speckles in interphase nuclei and generalized staining of condensedchromosomes in mitotic nuclei was observed.

Localization of DFS70 at the Ultrastructural Level

A high titered human anti-DFS70 was selected for immunoelectronmicroscopy and the targets were small pieces of mouse intestine fixed at4° C. with 1% glutaraldehyde as described in Materials and Methods.Mouse intestine was used as the substrate because p75 has been shown tobe ubiquitously expressed at the mRNA level in many different tissuesincluding small intestine (Ge et al., EMBO J. 1998; 17:6723-6729) andnumerous studies have shown that human autoantibodies react withcellular antigens which are highly conserved between mouse and man (Tan,Cell 1991; 67:841-842). Immunoelectron microscopy demonstrated thatDFS70/p75 was localized in interphase chromatin and appeared to beconcentrated over areas of condensed chromatin in the periphery of thenucleus and also in perinucleolar chromatin. The nucleolus and theinterchromatin regions contained relatively few colloidal goldparticles.

As shown in Table 1, 18 AD patients had what appeared to be acharacteristic DFS staining pattern by immunohistochemistry, but four ofthese sera (16,77,78,99) were negative in Western blotting againstrecombinant DFS70. It is possible that if these four sera did haveantibodies to DFS70/p75 transcription coactivator, the epitope(s)recognized by the four sera might not be primary sequence regions of theprotein but are conformation-dependent epitopes which are lost duringdenaturation in the polyacrylamide gel electrophoresis-Western blottingprocedure. Alternatively, immunohistochemistry might be more sensitivethan immunoblotting for this antigen-antibody system. It is also ofinterest that five AD patients which did not display the prototypicDFS70 staining pattern were found to have antibody to recombinant DFS70in Western blotting.

The DFS70/p75 autoantibody/autoantigen system showed the highestprevalence in three disease conditions, i.e., atopic dermatitis (30%),asthma (16%) and interstitial cystitis (9%). It has been well documentedthat AD is frequently associated with bronchial asthma and that childrenwith AD are more likely to develop asthma in later life than non-ADchildren (Cooper, J Invest Dermatol 1994; 102:128-137; Beltrani, JAllergy Clin Immunol 1999; 104:S87-S98). In addition to the fact thatANAs are present in higher than normal frequency in IC patients, thereare several reports that allergic disorders are a major symptomcomplaint in IC. In a recent study on 374 patients, food allergies werereported in 25.1% and hay fever in 24.9% (Kozial et al., J Urol 1993;149:465-469). Earlier studies have also pointed out the commonoccurrence of allergic disorders in IC and the role played by mast cells(Messing, et al. Urology 12:381-392; Hanno et al., J Urol 1990;143:278-281). None of these studies reported on the frequency of atopicdermatitis and it might be possible that in IC, some of the patientswith allergic diathesis might also have AD and that this could be acommon thread running through the three different disease conditions.

It is of interest that the putative function of the DFS70/p75 antigenhas been reported to be a transcription co-activator (Ge et al., EMBO J.1998; 17:6723-6729). There are two isoforms which are spliced productsof the same gene and encode for p75 as well as a truncated p52kilodalton protein which lacks the C-termninal region of p75. In studiesshowing that both these proteins functioned as transcriptionco-activators in an in vitro transcription system dependent on RNApolymerase II and general transcription factors, the shorter isoform p52was more active for most activation domains compared to p75. Humanautoantibodies recognized exclusively the p75 isoform, indicating thatthe epitope was located in the C-terminal region of p75 which is absentin p52. We and others have advanced the concept that spontaneouslyoccurring autoantibodies are antigen-driven (Tan, The Immunologist 1999;7:85-92; Radic et al., Ann Rev Immunol 1994; 12:487-520) and if this isthe case for the autoimmune response to DFS70, the immunogen which isdriving the immune reactin is the larger isoform, p75. At the presenttime, the significance of this finding is unknown, but although p75 is aless active transcription co-activator, it is possible that itsinvolvement in transcription might be related to certain specificactivation domains which were not used in the reported studies (Ge etal., EMBO J. 1998; 17:6723-6729). In this respect, future studies couldaddress the question whether p75 might enhance transcription activityfor certain genes predisposing to the development of atopy (Forrest etal., J Allergy Clin Immunol 1999; 104:1066-1070).

Our studies using autoantibodies from patients with AD and IC, andantibodies from rabbits immunized with p75 show that DFS70/p75 islocalized in the nucleoplasm by immunohistochemistry. This is consistentwith the function of p75 as a transcription co-activator associated withRNA pol II. Immunoelectron microscopy studies also show that DFS70 islocalized in the nucleus and preferentially in heterochromatin areas.This is in contrast to the reports by Singh et al. (Singh et al., InvestOphthalmol Vis Sci 1999; 40:1444-1451) on the same protein which theycall lens epithelium derived growth factor (LEDGF) which was initiallyreported to be a cytoplasmic protein (Singh et al., Invest OphthalmolVis Sci 1998; 39:3590 (Abst)) and subsequently reported to be present inthe cytoplasm of lens epithelial cells at 4°, 15° and 28° and in thenucleus and nucleolus at 37° (Singh et al., Invest Ophthalmol Vis Sci1999; 40:1444-1451). Its localization in the cytoplasm at temperatureslower than 37° is non-physiological and the significance of this isunknown. These authors identified LEDGF and isolated a cDNA encodingthis protein with the serum of a patient with age-related cataract. Someof the sera from patients with age-related cataract appeared to containantibodies to LEDGF. The interesting feature is that cataracts and otherocular complications have been reported in certain patients with AD(Nakano et al., J Jpn Ophthalmol Soc 1997; 101:64-68; Fagerholm et al.,Graefes Arch Clin Ophthalmol 1984; 221:149-152) and in one report, lensopacities were noted in 23.8% of 133 eyes examined (Nakano et al., J JpnOphthalmol Soc 1997; 101:64-68).

We have proposed the notion that spontaneously occurring autoantibodiesmight be regarded as messengers from the immune system that reportabnormal events which involve cellular components participating indisease-related mechanisms and that these cellular components might bethe antigens driving the immune response (Tan, The Immunologist 1999;7:85-92). The autoantibodies themselves might not be playing apathogenic role, but can be used to identify the cellular componentsparticipating in such disease processes. However, the question whichneeds to be addressed is whether the autoimune process in AD involvingDFS70/p75 is playing any role in pathogenesis. The current study doesnot address the question of the cell mediated immune system sincecellular infiltrates consisting of increased numbers of lymphocytes,monocytes, macrophages and mast cells are present in atopic lesions(Cooper, J Invest Dermatol 1994; 102:128-137; Leung, J Allergy ClinImmunol 1999; 104:S99-S108). An important study would be examination ofslin biopsies from AD lesions to determine whether there might beabnormal expression of DFS70/p75 mRNA and protein. Such studies couldnot be done since the present work was carried out only with serumspecimens and further studies along these lines are needed. We did findIgE autoantibodies to DFS70/p75 in both AD and IC patients and thisfinding lends some support to the possible pathogenic significance ofthe present findings in view of several studies showing prominence ofIgE positive Langerhans cells in AD lesions (Leung, J Allergy ClinImmunol 1999; 104:S99-S108).

1. A process of screening a patient for atopic dermatitis comprisingdetermining in sera of the patient the presence of antibodies against anantigen comprising a nuclear transcription co-activator p75, wherein thepresence of said antibodies indicates atopic dermatitis.
 2. The processof claim 1 wherein the patient is asthmatic.
 3. The process of claim 1wherein the patient has interstitial cystitis.
 4. The process of claim 1wherein the sera from the patient is contacted with and maintained for aperiod of time sufficient for formation of an immune complex betweenantibodies in the sera and the antigen.
 5. The process of claim 1wherein the antibodies are IgG molecules.
 6. The process of claim 1wherein the antibodies are IgE molecules.